Cholesterol detection reagent

ABSTRACT

An object of the present invention is to provide a novel cholesterol detection reagent comprising a substance which can specifically binds to cholesterol to detect it, and a method for detecting cholesterol using the reagent. The present invention provides a cholesterol detection reagent comprising a polyethylene glycol cholesteryl ether which may be labeled.

TECHNICAL FIELD

The present invention relates to a cholesterol detection reagent, and amethod for detecting cholesterol using the reagent. More specifically,the present invention relates to a cholesterol detection reagent whichcomprises a polyethylene glycol cholesteryl ether, and a method fordetecting cholesterol using the reagent.

BACKGROUND ART

The content and distribution of intracellular cholesterol is stringentlyregulated. Inside the cells, cholesterol is accumulated in the postGolgi membranes (M. S. Bretscher, et al., Science 261,1280-1.(1993)). Onthe plasma membrane, cholesterol forms microdomains together withsphingomyelin and glycosphingolipids (A. Rietveld, et al., BiochimBiophys Acta 1376,467-79.(1998) ; and R. E. Brown, J Cell Sci111,1-9.(1998)). Caveolins and other classes of proteins such asglycosylphosphatidylinositol (GPI)-linked glycoproteins and duallyacylated non-receptor tyrosine kinases are located in these domains (T.V. Kurzchalia, et al., Curr Opin Cell Biol 11,424-31.(1999) ; and E.Ikonen, et al., Traffic 1,212-7.(2000)). These domains are known aslipid rafts. Lipid rafts are postulated to play an important role incellular functions such as signaling, adhesion, motility, and membranetraffic (D. A. Brown, et al., Annu Rev Cell Dev Biol 14,111-36(1998);and K. Simons, et al., Nat Rev Mol Cell Biol 1,31-9.(2000)). Reductionof cellular cholesterol contents by removing surface cholesterol withmethl-β-cyclodextrin (M β CD) or by metabolic inhibitors results indisintegration of these domains (L. J. Pike, et al., J. Biol Chem273,22298-304.(1998) ; A. Pralle, et al., J Cell Biol148,997-1008.(2000) ; and K. Roper, et al., Nat Cell Biol2,582-92.(2000)).

Cellular content of cholesterol is controlled via the balance of de novosynthesis and exogenously obtained cholesterol through the endocytosisof lipoproteins (M. S. Brown, et al., Proc Natl Acad Sci USA96,11041-8.(1999) : K. Simons, et al., Science 290,1721-6.(2000) ; andY. A. Ioannou, Nat Rev Mol Cell Biol 2,657-68.(2001)). The collapse ofthis control leads to pathogenic conditions such as arteriosclerosis orNiemann-Pick type C (NPC) (P. G. Pentchev et al., Biochim Biophys Acta1225,235-43.(1994) ; and L. Liscum, Traffic 1,218-25.(2000)). Internalmembrane domains of late endosomes rich in lysobisphosphatidic acid areimplicated in regulation of cholesterol transport by acting as acollection and distribution device (T. Kobayashi et al., Nat Cell Biol1,113-8.(1999)). However, little is known about the intracellulartransport of cholesterol and/or cholesterol-rich membrane domains.

Poly(ethylene glycol)cholesteryl ethers (PEG-Chols) are an unique groupof nonionic amphiphatic molecules consisting of hydrophobic cholesteryland hydrophilic poly(ethylene glycol) moieties (FIG. 1A) (H. Ishiwata,et al., Biochim Biophys Acta 1359,123-35(1997)). When added to livingcells in culture, PEG(50)-Chol (moleculaw weight is 2587; 50 (inparentheses) is the number of ethylene glycol repeat) inhibitedclathrin-independent, caveolac-like endocytosis under the condition ofwhich clathrin-mediated internalization of transferrin was not affected(T. Baba et al., Traffic 2,501-12.(2001)). However, it remains unknownwhat type of cell components the PEG-Chol interacts with.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to identify a molecule to whicha polyethylene glycol cholesteryl ether can specifically bind in cells.Further, it is another object of the present invention to provide anovel cholesterol detection reagent comprising a substance which canspecifically binds to cholesterol to detect it, and a method fordetecting cholesterol using the reagent.

The present inventors have carried out intensive studies to achieve theaforementioned objects. Taking into consideration the previous findingsthat PEG(50)-Chol specifically inhibits clathrin-independentendocytosis, the present inventors have assumed that PEG-Chol canspecifically interact with one or more Lipid raft components, and haveconfirmed by overlay assay that PEG-Chol binds to various lipids invitro. Moreover, as a result of studies regarding a substance with whichPEG-Chol interacts in cells, the present inventors have found thatPEG-Chol can specifically bind to cholesterol. The present invention hasbeen completed based on these findings.

Thus, the present invention provides a cholesterol detection reagentcomprising a polyethylene glycol cholesteryl ether which may be labeled.

In another aspect of the present invention, there is provided a methodfor detecting cholesterol, wherein a polyethylene glycol cholesterylether which may be labeled is used.

In the present invention, it is preferable to use a polyethylene glycolcholesteryl ether, which is labeled with an affinity substance orfluorescent substance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an in vitro binding experiment usingPEG-Chol.

FIG. 2 shows the results of a labeling experiment with PEG-Chol usingcells. The bar indicates 20 μm.

FIG. 3 shows the results obtained by examining the distribution offPEG-Chol on the surface of cells.

FIG. 4 shows the results obtained by examining the distribution offPEG-Chol on the surface of cells.

FIG. 5 shows the results obtained by examining the distribution offPEG-Chol on the surface of cells.

FIG. 6 shows the results obtained by analyzing the intra-membranedistribution of cholesterol and the fate of cholesterol on the surfaceof cells.

FIG. 7 shows the results obtained by analyzing the intra-membranedistribution of cholesterol and the fate of cholesterol on the surfaceof cells.

FIG. 8 shows the results obtained by analyzing the intra-membranedistribution of cholesterol and the fate of cholesterol on the surfaceof cells.

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiments of the present invention will be described below.

The cholesterol detection reagent of the present invention comprises apolyethylene glycol cholesteryl ether, which may be labeled.

The polyethylene glycol cholesteryl ether used in the present inventionis a compound having the structure shown in FIG. 1A, which consists of ahydrophobic cholesteryl moiety and a hydrophilic polyethylene glycolmoiety (H. Ishiwata, et al., Biochim Biophys Acta 1359, 123-35 (1997)).In the structure, n represents the repeated number of ethylene glycolsin the polyethylene glycol moiety. The number of n in the polyethyleneglycol cholesteryl ether used in the present invention is notparticularly limited, as long as it does not affect adversely thebinding ability with cholesterol. For example, the number of n isbetween 10 and 1,000, preferably between 20 and 200, and more preferablybetween 20 and 100. An example of a preferably used compound may includea polyethylene glycol cholesteryl ether containing a polyethylene glycolmoiety where n=50.

The polyethylene glycol cholesteryl ether used in the present inventionis a known compound, which is, for example, described in theaforementioned publication (H. Ishiwata et al., Biochim Biophys Acta1359, 123-35 (1997)). The polyethylene glycol cholesteryl ether used inthe present invention can be produced by dissolving cholesterol in asolvent and injecting ethylene glycol gas into the obtained solution soas to perform a reaction (Ishiwata et al., Chem Pharm Bull 43, 1005-1011(1995)). Other than this method, the polyethylene glycol cholesterylether can also be produced by a method involving allowingtoluenesulfonate of cholesterol to react with polyethylene glycol (Patelet al., Biochim Biophys Acta 797: 20-26 (1984)).

As a polyethylene glycol cholesteryl ether used in the presentinvention, those to which a labeling substance used for detection bindsare preferably used. The type of such a labeling substance is notparticularly limited. Examples of such a labeling substance may includean affinity substance, a fluorescent substance, and a radioactivesubstance.

Examples of an affinity substance used herein may include biotin anddigoxigenin. Examples of a fluorescent substance used herein may includefluorescein, FITC, BODIPY 493/503, BODIPY FL, dialkylaminocoumarin,2′,7′-dichlorofluorescein, hydroxycoumarin, methoxycoumarin,naphthofluorescein, Oregon Green 514, tetramethylrhodamine (TMR),X-rhodamine, NBD, TRITC, Texas, Cy5, Cy7, IR144, FAM, JOE, TAMRA, andROX. Examples of a radioactive substance used herein may include ³²P,¹³¹I, ³⁵S, ⁴⁵Ca, ³H, and ¹⁴C. Other than these substances, oxidationstress-detecting agents such as carboxy-PTIO and DTCS (Dojin),NO-generating agents such as BNN5 (Dojin), various caged amino acids,chelating agents (e.g. DTPA, EDTA, NTA, etc.), and various carboxydisulfides (having the structure of (carboxylic acid) S—S (carboxylicacid)) may also be used.

The form of the cholesterol detection reagent of the present inventionis not particularly limited, as long as it contains the aforementionedpolyethylene glycol cholesteryl ether which may be labeled. The form maybe either a solid or a liquid (a solution, a suspension, etc.). Whencholesterol detection reagent is in the form of a liquid, thepolyethylene glycol cholesteryl ether is dissolved or suspended in asuitable solvent (which is preferably an organic solvent or the like,regarding which the polyethylene glycol cholesteryl ether exhibits acertain degree of solubility), so as to prepare the reagent. To thereagent of the present invention, which is provided in theaforementioned form, assistant agents other than the polyethylene glycolcholesteryl ether (e.g. a preservative, a stabilizer, a pH buffer, etc.)can also be added as appropriate.

The present invention also provides a method for detecting cholesterolusing the polyethylene glycol cholesteryl ether which may be labeled.Detection may be carried out in vitro, in a cell, or in vivo. First, aspecimen containing cholesterol to be detected is allowed to come intocontact with a polyethylene glycol cholesteryl ether (which ispreferably labeled) under certain conditions, so as to bind them to eachother.

After completion of the binding, the polyethylene glycol cholesterylether which was bound to cholesterol is detected. Detection canappropriately be carried out depending on the type of the label used.

When biotin is used as a label for example, detection can be carried outusing avidin or streptavidin, which specifically bind to biotin. Forexample, a biotin-labeled polyethylene glycol cholesteryl ether whichwas bound to cholesterol is allowed to react with avidin orstreptavidin, and a biotinated alkaline phosphatase is then allowed tobind thereto, so that the enzyme binds thereto via biotin. After anunbounded enzyme portion has been removed, nitroblue tetrazolium (NBT),which is a substrate of alkaline phosphatase, is allowed to react with5-bromo-4-chloro-3-indolylphosphate (BCIP). As a result, when abiotin-labeled polyethylene glycol cholesteryl ether exists, thedevelopment of a violet color is seen, and it can therefore be detected.When digoxigenin is used as a label, detection can be carried out usingan alkaline phosphatase-labeled anti-digoxigenin antibody by the samemethod as described above. Other than alkaline pbosphatase, a systemusing horseradish peroxidase has also been known as an enzyme used forcolor development.

When a fluorescent substance such as a fluorescein is used, apolyethylene glycol cholesteryl ether which was bound to cholesterol canbe detected by measuring fluorescence after completion of the reactionwith cholesterol. That is, fluorescence energy generated as a result ofapplication of a certain amount of excitation light is measured, so asto qualitatively or quantitatively detect fluorescence. Whenfluorescence is quantitatively detected, the intensity of fluorescenceenergy can be evaluated as an indicator of the abundance of cholesterol.Such fluorescence energy or fluorescence can be measured using asuitable detector or fluorescence microscope, which are commerciallyavailable.

When a radioactive substance is used, after completion of the reactionwith cholesterol, radioactivity which was bound to the cholesterol ismeasured by a method known to a person skilled in the art, so as todetect the cholesterol.

The present invention will be more specifically described in thefollowing examples. However, the examples are not intended to limit thescope of the present invention.

EXAMPLES EXAMPLE 1 In Vitro Binding Experiment using PEG-Chol

(Methods)

-   (1) The binding ability of biotinylated PEG-Chol (bPEG-Chol: one    molecule of biotin is conjugated to the terminal ethylene glycol    moiety of PEG(50)-Chol) (10 μM) to various amounts of lipids was    analyzed by overlay assay, which was performed on TLC plates, as    described in the previous report (K. Igarashi et al., J Biol Chem    270, 29075-8. (1995)). The results are shown in FIG. 1B.-   (2) The binding of bPEG-Chol (10 μM) to various lipids, glycolipids,    and cholesterol oleate (100 nmol) was examined in the same manner as    described in (1) above. The results are shown in FIG. 1C.    -   (3) The binding of bPEG-Chol to a mixture consisting of        glucosylceramide (GlcCer) and sphingomyelin (SM) or a mixture        consisting of glucosylceramide and dioleoylphosphatidylcholine        (DOPC) (total 30 nmol with the ratio indicated in FIG. 1D) was        analyzed. The results are shown in FIG. 1D.-   (4) The traces of thermograms obtained by differential scanning    calorimetry performed on GlcGer, SM, GlcCer+SM (1:1), and    GlcCer+DOPC (1:1) were measured. 500 μl of a suspension containing 1    mM liposomes (GlcCer, SM, and DOPC) or 2 mM liposomes (GlcCer+SM and    GlcCer+DOPC) was measured using MicroCal VP-DSC. The results are    shown in FIG. 1E.-   (5) The fluorescence image of a monolayer composed of a mixture    consisting of GlcCer and DOPC at a ratio of 1:1 was obtained. A    lipid monolayer was prepared by injecting 20 μl of a chloroform    solution of 1 mM GlcCer+DOPC containing 0.5% C12-BODIPY-PC    (Molecular Probes) into a USI system (Fukuoka, Japan) FSD-500    Langmuir-Blodgett trough. The C12-BODIPY-PC was preferentially    partitioned into the DOPC phase. The surface pressure was adjusted    to 10 mM/m. Using an Olympus Power BX fluorescent microscope    equipped with an LM Plan FI 50× objective and a Toshiba 3CCD camera,    a fluorescence image was recorded. The results are shown in FIG. 1F.    The bar indicates 50 μm.-   (6) Using 1 mM sphingomyelin vesicles containing various amounts of    cholesterols, the binding of fluorescein PEG-Chol (fPEG-Chol)    containing a fluorescein on the distal end of a PEG chain was    analyzed (H. Ishiwata et al., Biochim Biophys Acta 1359, 123-35    (1997)). Vesicles were produced in the manner described in the    previous report (A. Miyazawa et al, Mol Immunol 25, 025-31. (1988)).    Vesicles were incubated with fPEG-Chol at room temperature for 30    minutes. Unbounded fPEG-Chol was washed by centrifugation at 15 K×g    for 15 minutes. The fluorescence of the pellet was measured, and    normalized with phosphorus of sphingomyelin. The results are shown    in FIG. 1G.-   (7) Transfer of fPEG-Chol between membranes was analyzed. 500 μM    (final concentration) SM/Chol (1:1) liposomes were added to    liposomes (50 μM) composed of SM alone or SM/Chol (1:1), which    contained 0.5 μM fPEG-Chol and 0.5 μM    N-rhodamine-dipalmitoylphosphatidylethanolamine. The release of    fluorescence resonance energy transfer (FRET) was measured by    monitoring time course of fluorescence emission spectrum at 535 nm    with excitation at 488 nm. The results are shown in FIG. 1H.

It is to be noted that cholesterol and cholesterol oleate were purchasedfrom Sigma (St. Louis, Mo.). Galactosylceramide, glucosylceramide, andlactosylceramide were purchased from Matreya (State College,Pennsylvania). All other lipids were purchased from Avanti Polar lipids(Alabaster, Ala.).

Chol represents cholesterol, SM represents sphingomyelin, PC representsphosphatidylcholine, PS represents phosphatidylserine, PE representsphosphatidylethanolamine, PI represents phosphatidylinositol, PArepresents phosphatidic acid, GM1 represents ganglioside GM1, GM2represents ganglioside GM2, GM3 represents ganglioside GM3, GalCerrepresents galactosylceramide, GlcCer represents glucosylceramide, andLacCer represents lactosylceramide.

(Results)

Biotinylated PEG-Chol (bPEG-Chol: one molecule of biotin is conjugatedto the terminal ethylene glycol moiety of PEG(50)-Chol) was added tospots of various lipids. After washing, the binding was monitored byHRP-conjugated streptavidin using 4-chloro-1-naphtol as a substrate(FIGS. 1B and 1C) (A. Yamaji et al., J Biol Chem 273, 5300-6. (1998)).The bPEG-Chol bound to cholesterol and neutral glycolipids (e.g.galactosylceramide, glucosylceramide (GlcCer), and lactosylceramide).However, the bPEG-Chol did not bind to phospholipids and acidicglycolipids (gangliosides) tested. Also, it did not bind to cholesterylester and cholesterol oleate. Moreover, the addition of sphingomyelin(SM) abolished the binding of bPEG-Chol to glucosylceramide, butsphingomyelin (SM) did not have such effects ondioleoylphosphatidylcholine (DOPC) (FIG. 1D).

Differential scanning calorimetry (DSC) showed that an equimolar mixtureconsisting of SM and GlcCer gave a gel-to-liquid crystalline phasetransition temperature in the middle of those of SM and GlcCer (FIG.1E). In contrast, the phase transition temperature of an equimolarmixture consisting of DOPC and GlcCer was very close to that of GlcCer,whereas the phase transition temperature of DOPC was much lower thanthat of SM. These results suggest that GlcCer is miscible with SMwhereas a binary mixture consisting of this lipid and DOPC is segregatedin different domains.

In order to confirm that GlcCer is segregated from DOPC, a monolayersystem was employed (FIG. 1F). A monolayer experiment clearly showedthat GlcCer (black) was segregated from DOPC (green) to form domains atan air-water interphase. These results suggest that PEG-Chol binds toneutral glycolipids only when they are clustered each other. Thedetergent solubility of cell membranes (D. A. Brown et al., Cell 68,533-44. (1992)) and the measurement of lipid partitioning in modelmembranes (T. Y. Wang et al., Biophys J 79, 1478-89. (2000)) suggestthat glycolipids are distributed to sphingomyelin-rich membranes incells. Taking into account the high concentration of sphingomyelin inbiomembranes, these results suggest that PEG-Chol may not significantlybind to glycolipids in cells. In contrast to glycolipids, the additionof sphingomyelin did not affect bPEG-Chol binding to cholesterol untilthe cholesterol content was reduced to less than 10%.

In order to examine the binding of PEG-Chol to cholesterol, a liposomeexperiment was further conducted using fluorescein PEG-Chol (fPEG-Chol)containing a fluorescein on the distal end of a PEG chain. As in thecase of overlay assay, the addition of cholesterol increased the bindingof fPEG-Chol to sphingomyelin liposomes (FIG. 1G). The fact thatfPEG-Chol did not bind to SM liposomes when the cholesterol content waslow (10%) suggests that fPEG-Chol recognizes cholesterol-rich domains inthe aforementioned membranes.

PEG-Chol is water-soluble and can be transferred between membranes. InFIG. 1H, the transfer of fPEG-Chol between membranes was measured. Inorder to measure the transport of fPEG-Chol, fluorescence resonanceenergy transfer (FRET) between fPEG-Chol and rhodamine-labeledphosphatidylethanolamine (rhodamine-PE) used as a non-exchangeablemarker was measured (J. W. Nichols et al., Biochemistry 21, 1720-6.(1982)). In donor liposomes, fPEG-Chol fluorescence was quenched byFRET. However, once fPEG-Chol was transported to acceptor liposomes,fluorescence was de-quenched. When SM liposome was used as a donor andSM/Chol (1:1) liposome was used as an acceptor, the efficient transportof fPEG-Chol was observed. In contrast, when both donor and acceptorwere SM/Chol (1:1), fPEG-Chol did not transfer significantly. Theseresults indicate that PEG-Chol is preferentially incorporated intocholesterol-rich membranes, and that once it is incorporated therein, itis trapped in the membranes.

Example 2 Labeling Experiment using Cells Labeled with PEG-Chol

(Methods)

As described in the previous report (T. Kobayashi et al., Nat Cell Biol1, 113-8. (1999)), normal (FIGS. 2A to 2D) and NPC (FIGS. 2E to 2H)human skin fibroblasts were fixed and permeabilized. Cells were thentriply labeled with 5 μM fPEG-Chol (FIGS. 2A and 2E), 50 μg/ml filipin(FIG. 2B and 2F), and an anti-TGN 46 antibody (Serotec Inc., Oxford,U.K.) (FIGS. 2C and 2G). The specimens were observed using a Zeiss LSMconfocal microscope. FIGS. 2D and 2H show merged images. White colorindicates the co-localization of 3 types of fluorophores. With regard tothe specimens stained with fPEG-Chol and filipin, normal cells and NPCcells were exposed to the laser light differently since the fluorescenceis much brighter in NPC cells.

In FIG. 2I and 2J, NPC cells were allowed to grow in the presence ofnormal serum (FIG. 2I) or delipidated serum (FIG. 2J). Thereafter, thecells were permeabilized and labeled with fPEG-Chol.

In FIG. 2K and 2L, NPC skin fibroblasts were fixed and permeabilized.Thereafter, the cells were labeled with fPEG-Chol in the presence of 1mM sphingomyelin liposomes (FIG. 2K) or sphingomyelin/cholesterol (1:1)liposomes (FIG. 2L).

In FIGS. 2M to 2R, a melanoma cell line MEB4 (FIGS. 2M to 2O) and amutant GM95 that is a melanoma cell line defective in glycolipidsynthesis (FIGS. 2P to 2R) were fixed and permeabilized. Thereafter, thecells were doubly labeled with fPEG-Chol (FIGS. 2M and 2P) and filipin(FIGS. 2N and 2Q). Similar fluorescence pattern in MEB4 and GM95suggests that the labeling with fPEG-Chol is not primarily dependent onglycolipids. fPEG-Chol labeling was co-localized with filipin labeling(FIGS. 2O and 2R).

(Results)

The in vitro interaction of PEG-Chol and various lipids suggests thatthis molecule will be incorporated into specific cholesterol-richmembranes or membrane domains in the cell, When fPEG-Chol was added topermeabilized human skin fibroblasts, the Golgi apparatus emitted brightfluorescence (FIG. 2A). A similar but less clear pattern of fluorescencehad previously been observed when filipin forming a complex withcholesterol had been used (J. Sokol et al., J Biol Chem 263, 3411-7.(1988); and T. Kobayashi et al., Nat Cell Biol 1, 113-8. (1999)).fPEG-Chol staining was partially co-localized with a trans-Golgi networkmarker, TGN46 (A. R. Prescott et al., Eur J Cell Biol 72, 238-46.(1997)). Incomplete overlap suggests that TGN46 and cholesterol aredifferently distributed in the Golgi apparatus. Niemann-Pick type C(NPC) is an autosomal recessive, neurovisceral disease. The hallmark ofthe NPC syndrome is the intracellular accumulation of unesterifiedcholesterol (P. G. Pentchev et al., Biochim Biophys Acta 1225, 235-43.(1994); L. Liscum, Traffic 1, 218-25. (2000); and T. Kobayashi et al.,Nat Cell Biol 1, 113-8. (1999)). Differing from normal fibroblasts,fPEG-Chol stains perinuclear vesicles as well as the Golgi apparatus inNPC fibroblasts (FIG. 2E). In this case also, the fluorescence wasco-localized with filipin (FIGS. 2F and 2H).

Cholesterol accumulation was significantly decreased when NPC cells wereallowed to grow in the absence of lipoproteins (J. Sokol et al., J BiolChem 263, 3411-7. (1988)). When NPC cells were allowed to grow in thepresence of delipidated serum instead of normal serum, perinuclearlabeling with fPEG-Chol was dramatically decreased (FIGS. 2I and 2J).When fPEG-Chol was preincubated with SM/Chol (1:1) liposomes, fPEG-Chollabeling was abolished (FIG. 2L). Cholesterol-free sphingomyelinliposomes showed much fewer effects under the same conditions (FIG. 2K).Once incorporated in membrane domains, fPEG-Chol was not removedtherefrom even using SM/Chol liposomes. This strengthens the idea thatfPEG-Chol is trapped in cholesterol-rich membrane domains in the cell.

GM95 is a melanoma cell line defective in glycolipid synthesis (S.Ichikawa et al., Proc Natl Acad Sci USA 91, 2703-7. (1994)). In order toexamine the effects of glycolipids on PEG-Chol staining, GM95 wascompared with parent MEB4 cells. Both GM95 and MEB4 were labeled withfPEG-Chol in similar manners (FIGS. 2M and 2P). In addition, thislabeling was co-localized with filipin labeling. These results suggestthat the labeling of cells with fPEG-Chol was primarily dependent oncellular cholesterol but not on glycolipids.

Example 3 Distribution of fPEG-Chol on Cell Surface

(Methods)

Normal human skin fibroblasts were incubated together with cholera toxinlabeled with 1 μM fPEG-Chol and 5 μM AlexaFluor 594 at room temperaturefor 90 seconds. Thereafter, the cells were fixed with paraformaldehydefor 10 minutes. FIGS. 3A and 3C show fPEG-Chol fluorescence, and FIGS.3B and 3D show AlexaFluor 594 fluorescence. Small arrows indicatestructure, which were double-labeled with fPEG-Chol and cholera toxin.Large arrows indicate those labeled only with fPEG-Chol. Arrowheadsindicate the spots that are positive with cholera toxin alone. In FIGS.3E and 3F, before fixation, the cells were treated with (E) and without(P) 10 mM MβCD at 37° C. for 30 minutes. Thereafter, the cells werelabeled with 1 μM fPEG-Chol. In FIG. 3, the bar indicates 4 μm.

In FIGS. 4G to 4L, normal skin fibroblasts were labeled with 2 μMfPEG-Chol. Thereafter, the cells were incubated with a 5 μg/mlbiotinylated epidermal growth factor (EGF) at 4° C. for 20 minutes(FIGS. 4G and 4H), or at 37° C. for 2 minutes (FIGS. 4I and 4L).Thereafter, the cells were fixed with PBS containing 3% PFA and 8%sucrose, quenched, and then incubated with TRITC-labeled streptavidin at4° C. for 20 minutes. The specimens were observed with a Nikon TE 300microscope equipped with a Hamamatsu C-4742-98 cooled CCD camera. InFIG. 4, G and I indicate fPEG-Chol fluorescence, and H and J indicateAlexaFluor 594 EGF-fluorescence. In K and L in FIG. 4, the cells weredoubly labeled with 1 μM fPEG-Chol and an AlexaFluor 594-labeled choleratoxin B subunit prior to being stimulated by non-labeled EGF. In FIG. 4,K indicates fPEG-Chol fluorescence, and L indicates cholera toxinfluorescence. In FIG. 4, the bar indicates 4 μm.

In M to P in FIG. 5, B cell line A20.2J was incubated at 37° C. for 1minute without antibodies. Cells were then washed and fixed with 1% PFAfor 30 minutes, and then labeled with 0.7 μM fPEG-Chol and a 10 μg/mlAlexa 546-conjugated cholera toxin B subunit in 0.1% BSA on ice for 45minutes. After washing, the stained cells were observed under a ZeissLSM 510 confocal microscope. In FIG. 5, M indicates fPEG-Chol labeling,N indicates cholera toxin labeling, O indicates a merged image, and Pindicates a phase contrast image. Under these conditions, fPEG-Cholpermeates the fixed cells, so as to stain intracellular membranes aswell as plasma membranes. In contrast, cholera toxin did not enter thecells, and thus, it stained only the cell surfaces.

In Q to T in FIG. 5, A20.2J cells were stimulated with 15 μg/ml F(ab′)₂goat antibodies specific for mouse IgG+IgM (F(ab′)₂ anti-Ig) at 37° C.for 1 minute. Thereafter, the cells were fixed and stained as describedabove. In FIG. 5, Q indicates fPEG-Chol labeling, R indicates choleratoxin labeling, S indicates a merged image, and T indicates a phasecontrast image.

(Results)

In Example 3, the distribution of fPEG-Chol on the cell surface wasexamined (FIGS. 3 to 5).

Normal human skin fibroblasts were treated with fPEG-Chol, and thenwashed and fixed. Non-uniform surface labeling with higher fluorescencewere observed in small domains (with diameters between 200 and 500 nm)(FIG. 3A and 3C). Some of these domains were co-localized with anAlexaFluor 594-labeled cholera toxin B chain (FIGS. 3B and 3D). Choleratoxin binds to GM1, which is non-randomly distributed on the plasmamembranes and accumulates in caveolae (R G. Parton, J Histochem Cytochem42, 155-66. (1994)). When the cells were pretreated withmethyl-p-cyclodextrin (MβCD), which specifically removes cholesterolfrom cells, fPEG-Chol staining disappeared (FIG. 3E and 3F) (G. H.Rothblat et al., J Lipid Res 40, 781-96. (1999)).

Subsequently, the distribution of fPEG-Chol when cells were notstimulated with an epidermal growth factor (EGF) was measured. An EGFreceptor localized to cholesterol-rich plasma membrane domains, andthus, it was suggested that the binding of EGF to the EGF receptor isdependent on cell surface cholesterol (M. G. Waugh et al., Biochem SocTrans 29, 509-11. (2001): K. Roepstorff et al., J Biol Chem 8, 8 (2002);and T. Ringerike et al., J Cell Sci 115, 1331-40. (2002)). fPEG-Cholfluorescence was co-localized with the distribution of biotin-labeledEGF, when EGF was added at 4° C. (FIGS. 4G and 4H). When EGF was addedat 37° C., the clustering of EGF receptors was observed (FIG. 4J). Theseclusters were labeled with fPEG-Chol (FIG. 4I). The cell surfacedistribution of GM1 was also examined under these conditions. GM1 wasalso enriched in these clusters and further co-localized with fPEG-Chol(FIGS. 4K and 4L). These results indicate that EGF inducesre-distribution of both cholesterol and GM1 to the same clusters whereEGF receptors were enriched.

Re-distribution of plasma membrane ganglioside occurs during thecross-linking of B cell antigen receptors on the plasma membrane of a Bcell line A20.2J (M. J. Aman et al., J Biol Chem 276, 46371-8. (2001)).Whether or not fPEG-Chol is re-distributed by treatment with F(ab′)₂anti Ig was examined. Before the treatment, both AlexaFluor 594-labeledcholera toxin and fPEG-Chol outlined the entire surface (FIGS. 5M to5P). However, after stimulation with a F(ab′)₂ fragment for 1 minute,cholera toxin was accumulated in aggregated structures on the plasmamembranes (FIG. 5R). fPEG-Chol also localized to these structures (FIGS.5Q and 5S). These results indicate that cholesterol is re-distributedtogether with GM1 during stimulation of B cell lines.

Example 4 Analysis on Intra-Membrane Distribution of Cholesterol andFate of Cell Surface Cholesterol

(Methods)

-   (1) As described above, the plasma membranes of normal (FIG, 6A) and    NPC (FIG. 6B) fibroblasts were permeabilized using streptolysin O.    The cells were incubated with fPEG-Chol at room temperature for 30    minutes before washing and taking fluorescence images under a Zeiss    LSM 510 confocal microscope. The results are shown in FIG. 6.-   (2) Normal (FIGS. 7C to 7H) and NPC (FIGS. 7I to 7N) fibroblasts    were incubated with 1 μM fPEG-Chol at room temperature for 5    minutes. Cells were washed and incubated for 10 minutes (FIG. 7, F,    L and L), 60 minutes (FIG. 7, D, G, J, and M), and 180 minutes (FIG.    7, E, H, K, and N) at 37° C. in the presence of 1 mg/ml rhodamine    dextran. The results are shown in FIG. 7.-   (3) NPC fibroblasts were incubated with 1 μM fPEG-Chol at room    temperature for 5 minutes. Cells were then washed and incubated at    37° C. for 30 minutes (FIG. 8O). NPC fibroblasts were incubated with    1 μM fPEG-Chol at 4° C. for 30 minutes. Cells were then washed and    photographed. Cells were then washed and incubated at 37° C. for 30    minutes (FIG. 8P). NPC fibroblasts were treated with 5 μg/ml    brefeldin A for 30 minutes (FIG. 8Q), 5 82 g/ml nocodazole for 90    minutes (FIG. 8R), or 5 μg/ml cytochalasin B for 30 minutes (FIG.    8S) before incubation with 1 μM fPEG-Chol and 1 mg/ml rhodamine    dextran for 30 minutes. In FIG. 8T, NPC fibroblasts were incubated    with 1 μM fPEG-Chol for 30 minutes before treatment with 5 μg/ml    cytochalasin B for 30 minutes. In FIGS. 6 to 8, the bar indicates 20    μm.    (Results)

Little has been known about the intra-membrane distribution ofcholesterol. In the present example, whether or not cholesterol islocated in the cytoplasmic side or luminal side of the intracellularmembranes was examined by using semi-permeable cells. Plasma membranesof normal and NPC skin fibroblasts were selectively permeabilized bybacterial toxin streptolysin O. Cells were then incubated with fPEG-Chol(FIGS. 6A and 6B). The fPEG-Chol staining was dramatically differentfrom those obtained in fixed and permeabilized cells (FIGS. 2And 2E). Inaddition, there was a big difference between normal and NPC cells. Innormal skin fibroblasts, peripheral vesicle-like structures werestrongly stained, whereas in NPC cells, meshwork-Like structures werevisualized. These structures were not observed after cells were fixedand permeabilized, suggesting that these compartments were eitherfragile or detergent sensitive. Golgi apparatus and lateendosomes/lysosomes were not significantly labeled under theseconditions. These results suggest that cholesterol resides only in thelumen of these organelles. In contrast, peripheral vesicles in normalfibroblasts and meshwork structures in NPC cells contain cholesterol inthe cytoplasmic membranes.

The detailed mechanism(s) of the intracellular accumulation of freecholesterol in NPC cells is not well understood. Recent studies suggestthat the accumulation results from an imbalance in the brisk flow ofcholesterol among membrane compartments (Y. Lange et al., J Biol Chem275, 17468-75. (2000)). Both the endogenously synthesized cholesteroland that derived via LDL once reach the plasma membrane, they are theninternalized in the cell. Cruz et al. suggested that NPC1 (that is aprotein encoded by the gene whose mutation is responsible for thedisease) is involved in a post-plasma membrane cholesterol-traffickingpathway (J. C. Cruz et al., Biol Chem 275, 4013-21. (2000)). In order tochase the fate of cell surface cholesterol, filipin is not suitablebecause of the toxicity. A fluorescent cholesterol analog,dehydroergosterol, was shown to be endocytosed and accumulated inrecycling compartment in a CHO cell line (S. Mukherjee et al., Biophys J75, 1915-25. (1998); and M. Hao et al., J Biol Chem 277, 609-17.(2002)). DHE differs from cholesterol in having three additional doublebonds and an extra methyl group. Recently, it has been shown thatperfringolysin O binds selectively to cholesterol-rich membrane domains(A. A. Waheed et al., Proc Natl Acad Sci USA 98, 4926-31. (2001); and W.Mobius et al., J Histochem Cytochem 50,43-55. (2002)). Advantages ofusing fPEG-Chol may include higher stability and quantum efficiency ofthe fluorophore, lower background staining, lower cell toxicity, andpossibly minor structural perturbation at the working concentrationbecause of the relatively small size.

The fate of cell surface fPEG-Chol of normal fibroblasts was comparedwith that of NPC fibroblasts (FIGS. 7C to 7N). In the presentexperiment, 1 μM fPEG-Chol was used. This concentration of fPEG-Chol didnot affect the endocytosis of dextran and cholera toxin in this system.Cells were incubated with fPEG-Chol at room temperature for 5 minutes,washed, and further incubated at 37° C. in the presence of 1 mg/mlrhodamine dextran. In normal fibroblasts, cell surface was stronglylabeled after 5 minutes of fPEG-Chol labeling. Most of the fluorescencestayed on the plasma membrane after 10 minutes of chase (FIGS. 7C and7F). After 60 minutes of chase, nucleus became recognized as anon-labeled organelle surrounded by cytoplasmic fluorescent compartments(FIG. 7D). The overall pattern of these compartments was similar to thatdetected by DHE-MβCD in CHO cells (M. Hao et al., J Biol Chem 277,609-17. (2002)). However, fPEG-Chol also stained intracellular vesicles.Most of these vesicles were not co-localized with internalized rhodaminedextran (FIG. 7G). These vesicles are often observed in the periphery ofcells, like those observed in FIG. 6A. After 180 minutes, Golgiapparatus was prominently labeled with fPEG-Chol while rhodaminefluorescence was distributed in endosomes/lysosomes (FIGS. 7E and 7H).The fate of fPEG-Chol was dramatically different in NPC fibroblasts.After 10 minutes of chase, fPEG-Chol stained characteristic meshworkstructures (FIGS. 7I and 7L), which was never observed in normal cells.Even after 180 minutes of chase, most of the fPEG-Chol was retained inthis structure and Golgi fluorescence was hardly visible (FIGS. 7J and7M). Sometimes, internalized rhodamine dextran was surrounded by themeshwork structures (FIG. 7M, arrows), suggesting that these structureshave characteristics of endocytic compartments. These structures arevery similar to those observed in FIG. 6B.

The incorporation of fPEG-Chol into the meshwork structure istemperature dependent. At 4° C., fPEG-Chol stayed on the plasma membraneand was not incorporated into the meshwork (FIG. 8P). FIG. 8P alsoindicates that fPEG-Chol does not undergo spontaneous transbilayermovement. The fluorophores, which undergo spontaneous flip-flop, stainintracellular membranes under these conditions (R. E. Pagano et al., JCell Biol 91, 872-7. (1981); and R. E. Pagano et al., J Biol Chem 260,1909-16. (1985)). Subsequently, the internalization of fPEG-Chol andrhodamine dextran was measured in the presence of inhibitors. BrefeldinA (an inhibitor of post-Golgi transport and nocodazole, which inhibitsmicrotubule assembly) did not significantly affect the incorporation offPEG-Chol into meshwork. In contrast, meshwork structure was disappearedby cytochalasin B (which inhibits actin polymerization). Cytochalasin Bdid not affect the internalization of rhodamine dextran. In FIG. 8T,cells were labeled with fPEG-Chol before treatment with cytochalasin B.In this case also, the meshwork structure was disappeared, suggestingthat the meshwork structure is dependent on action network.

INDUSTRIAL APPLICABILITY

From the aforementioned results of the examples, it was demonstratedthat fPEG-Chol is a useful means for visualizing cholesterol-richdomains. That is to say, the present invention provides a novelcholesterol detection reagent having advantages such as higher stabilityand quantum efficiency of the fluorophore, lower background staining,lower cell toxicity, and possibly minor structural perturbation at theworking concentration because of the relatively small size.

1. A cholesterol detection reagent comprising a polyethylene glycolcholesteryl ether which may be labeled.
 2. The cholesterol detectionreagent according to claim 1 wherein the polyethylene glycol cholesterylether is labeled with an affinity substance or fluorescent substance. 3.A method for detecting cholesterol, wherein a polyethylene glycolcholesteryl ether which may be labeled is used.
 4. The method fordetecting cholesterol according to claim 3 wherein a polyethylene glycolcholesteryl ether which is labeled with an affinity substance orfluorescent substance is used.